Project 5 Project Summary Abstract The goal of this research program is to determine the effects of the EBV oncoproteins, latent membrane protein 1 (LMP1) and latent membrane 2 (LMP2) on the cellular proteome. Both LMP1 and LMP2 interact with ubiquitin ligases and likely modulate both the levels and location of cellular proteins. This proposal is based on the hypothesis that critical biologic properties of these two viral oncogenes are based on their effects on the cellular proteome. The proposed experiments will primarily focus on two specific biologic effects of LMP1 and LMP2A. We have previously shown that LMP1 affects the content of cellular exosomes which are internalized and activate growth stimulating signaling pathways in recipient cells. The contribution of the specific LMP1-interacting ubiquitin ligases to this process will be determined using LMP1 mutants and inhibition of the interacting ligases. The effects of LMP1 and specific mutants within its signaling domains on exosome quantity and composition will be determined using mass spectrometry. The requirement for proteins that we have identified as increased by LMP1 will be assessed using shRNAs or dominant negative forms to inhibit their expression or function and the protein composition of the LMP1-modulated exosomes will be determined. The effects on the biologic properties of the exosomes produced by the LMP1 mutants or in the absence of specific cellular proteins will be identified. LMP2A also affects cell growth and contributes to cell survival. In epithelial cell lines, LMP2 inhibits differentiation, increases migration, and can induce anchorage independence. During this last funding period we have determined that in transgenic mice, LMP2 enhances growth promotion by LMP1 resulting in increased development of carcinomas. Three major signaling motifs have been identified within LMP2 that interact with src family kinases, syk family kinases, and ubiquitin ligases. We have recently shown that the apoptosis induced by anchorage loss (anoikis) is inhibited by LMP2A. This inhibition was dependent on the induction of autophagy and required the ubiquitin ligase binding domain. We have also determined that LMP2 affects the expression levels of multiple proteins involved in vesicle transport. The contribution of specific LMP2 domains on cellular protein expression and localization will be determined using mass spectrometry of vesicular fractions and immunofluorescent staining. The requirement for induction of autophagy in LMP2-mediated growth effects will be determined.